The existing methods for creating therapeutic monoclonal antibodies are primarily hybridoma, phage display, B cell isolation and antibody sequence rescue, followed if necessary by humanization of the antibody. The humanization step can be eliminated by using transgenic animals which are capable of themselves generating human antibodies.
The hybridoma method involves isolating large numbers of B cells from an immunized organism which are then fused with a myeloma tumor cell. These cells are then screened for individual cells producing antibodies with the desired properties. Once a particular hybridoma of interest is detected, a cDNA encoding the antibody is isolated and sequenced by standard molecular biology techniques, followed by a process of humanization. Humanization is the process of placing the variable region of the antibody, usually from a mouse or rabbit, into a human antibody backbone so that the resulting molecule is less immunogenic when used as a therapeutic agent in a human host. Hybridoma technology is limited by its low efficiency; the need for large numbers of B cells, which are generally obtained by sacrificing mice and harvesting from them splenic B cells; and the relative instability of the hybridomas themselves.
Phage display is a technology that uses a library of recombinant phage which present on their surface randomly paired variable domains of heavy and light chain antibodies taken from human donors. The phages are panned against the antigen of interest and those which specifically bind are amplified and the process is repeated. After multiple rounds of panning, representative phages' DNA are isolated and sequenced. The variable domains are then transferred into Ig constant region backbones. This technology often requires artificial affinity maturation in order to generate antibodies of sufficient affinity, which involves making a series of point mutations and retesting the affinity of the resulting antibodies. Moreover, the panning process is quite laborious and is often performed using robotics.
B cell isolation and antibody sequence rescue techniques involve isolating individual or small numbers of B cells and culturing them in vitro in order to create small amounts of conditioned supernatants which can be used to screen for antibodies with the desired properties. The selected lymphocyte antibody method (SLAM) is one aspect of this technology. Once an antibody with the desired properties is detected, the antibody's heavy and light chain are recovered from the cell or cells by standard molecular techniques such as PCR, and the resulting antibody is then humanized in the same manner as a hybridoma-derived antibody.
The methods currently available for the generation of monoclonal antibodies are laborious, time consuming, and subject to inaccuracy. Thus, there remains a need in the art for an efficient, easy, and accurate method for generating monoclonal antibodies.